Plant-based biopharmaceuticals have gained a lot of interest in the past decade due to their reduced cost and relative safety compared to mammalian cell cultures. While the first plant-made recombinant ... [more ▼]

Plant-based biopharmaceuticals have gained a lot of interest in the past decade due to their reduced cost and relative safety compared to mammalian cell cultures. While the first plant-made recombinant proteins are now reaching the market, the production systems still need improvements to maximize their competitiveness. Optimizing production hosts requires the identification and subsequent inhibition of the most active endogenous peptidases, proteolysis being one of the main factors limiting yields. The aim of our study was to identify root-secreted proteases of Arabidopsis thaliana involved in target protein degradation (BSA) and inhibit them in vivo. Biochemical analyses identified serine proteases as the main class responsible for BSA degradation. An RT-qPCR experiment led to the choice of the serine protease gene SBT4.12 and its homologs as targets for an amiRNA-mediated silencing approach. Arabidopsis amiRNA-expressing lines showed lower levels of expression for SBT4.12 and reduced proteolytic activity in their rhizosecreted extracts. Crossing these lines with recombinant protein producing lines could lead to an improved production platform for proteins of interest. [less ▲]

Plant-based biopharmaceuticals have gained a lot of interest in the past decade due to their reduced cost and relative safety compared to mammalian cell cultures. While the first plant-made recombinant ... [more ▼]

Plant-based biopharmaceuticals have gained a lot of interest in the past decade due to their reduced cost and relative safety compared to mammalian cell cultures. While the first plant-made recombinant proteins are now reaching the market, the production systems still need improvements to maximize their competitiveness, proteolysis being one of the main factors limiting the yields. Identifying and inhibiting in vivo endogenous proteases involved in the degradation of recombinant proteins could then lead to a significant increase in production yields. In this study, we focused on two different production systems in Arabidopsis thaliana: rhizosecretion and cell suspensions. Extracellular proteases of both systems were used in vitro to study the conditions of target protein degradation (Bovine Serum Albumine, BSA). First, proteases from both systems degrade BSA at both acidic and neutral-to-basic pH conditions. Then, serine and metallopeptidases were shown to be the main protease classes responsible for BSA degradation by rhizosecreted proteomes or extracellular cell culture media, respectively. Finally, the biochemical tests were coupled to a bioinformatics analysis of publicly available transcriptomic data, in order to reduce the number of the proteases most likely involved in BSA degradation. Using this method, only five serine proteases and two metallopeptidases remain candidates for an amiRNA-mediated in vivo inhibition. [less ▲]

Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now ... [more ▼]

Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion), in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes) were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA) and human serum immunoglobulins G (hIgGs). Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing. [less ▲]

Proteases are involved in many physiological processes during the whole life of the plant, such as embryonic development, defense against pathogens, nutrition or mycorrhiza creation. However, the ... [more ▼]

Proteases are involved in many physiological processes during the whole life of the plant, such as embryonic development, defense against pathogens, nutrition or mycorrhiza creation. However, the functions of many of the 800 proteases of Arabidopsis thaliana still remain unknown. Besides discovering new functions, studying proteases can also result in improving plant biotechnology. Indeed, plants can be used as hosts for recombinant protein production. Some proteins of interest require to be secreted in order to fold properly, but production yields are limited due to their degradation by endogenous extracellular proteases. The aim of our study is to identify active root-secreted proteases of Arabidopsis thaliana. Their activity was first analyzed by in vitro incubation with a target protein (BSA) at different values of pH and in the presence of proteases inhibitors. This analysis identified serine proteases as the major protease class involved in BSA degradation. Then, an Activity-Based Protein Profiling approach led to the labeling of two active serine proteases in the root-secreted sample. Finally, a further step towards the identification by mass spectrometry, based on affinity purification, was developed. [less ▲]

Besides traditional production systems, such as bacteria, yeasts and mammal cells, plants can now be used to produce eukaryotic recombinant proteins. Their advantages as hosts for protein production ... [more ▼]

Besides traditional production systems, such as bacteria, yeasts and mammal cells, plants can now be used to produce eukaryotic recombinant proteins. Their advantages as hosts for protein production include correct post-translational modifications, low cost of maintenance and no risk of contamination by human pathogens. Targeting heterologous proteins to the extracellular space is required for the correct folding of complex proteins and makes harvesting and purification easier. However, the quantity and the quality of recombinant proteins have been proved to be reduced by the action of endogenous co-secreted proteases. In this study, we aimed at identifying active root-secreted (rhizosecreted) proteases in the model plant Arabidopsis thaliana. Their activity was assayed by in vitro degradation of a target protein (Bovine Serum Albumine, BSA) in a range of pH. The protease classes involved in BSA degradation were evaluated by inhibitor-based assays that revealed serine proteases as the major class involved in this degradation in any tested conditions. As a first step towards identification, and subsequent silencing, of the most active members of this class, rhizosecreted proteases are being analyzed by the “Activity-Based Protein Profiling” approach. [less ▲]

Besides traditional production systems, such as bacteria, yeasts and mammal cells, plants can now be used to produce eukaryotic recombinant proteins. Their advantages as hosts for proteins production ... [more ▼]

Besides traditional production systems, such as bacteria, yeasts and mammal cells, plants can now be used to produce eukaryotic recombinant proteins. Their advantages as hosts for proteins production include correct post-translational modifications, low cost of maintenance and no risk of contamination by human pathogens. Targeting heterologous proteins to the extracellular space is required for the correct folding of complex proteins and makes harvesting and purification easier. However, the quantity and the quality of recombinant proteins have been proved to be reduced by the action of endogenous co-secreted proteases. In this study, we characterized root-secreted proteases in the model plant Arabidopsis thaliana, at the activity and expression levels. Their activity was analyzed by in vitro degradation of a target protein (Bovine Serum Albumine, BSA) in a range of pH and in the presence of several proteases inhibitors. Serine proteases were identified as the major protease class involved in the degradation of BSA under all tested conditions. As a first step towards the identification of the key players, the expression level of selected members of this class was analyzed by quantitative RT-PCR in roots and leaves. [less ▲]

Plants are promising hosts for the production of complex recombinant pharmaceuticals, such as antibodies (mAbs), because they offer an inexpensive and safer alternative to traditional production systems ... [more ▼]

Plants are promising hosts for the production of complex recombinant pharmaceuticals, such as antibodies (mAbs), because they offer an inexpensive and safer alternative to traditional production systems. The plant-based production of mAbs, which are multimeric glycoproteins, require their targeting to the secretory pathaway where they are properly folded and matured. However, co-secretion of endogenous proteases, which can represent up to 10% of the extracellular proteins (secretome), is known to significantly alter the yield and quality of secreted mAbs. In this study, we analyzed the proteolytic activities in root-secretome of Arabidopsis thaliana and Nicotiana tabacum. Root-secretomes were recovered by salt extraction and the protease activity was assayed in vitro or by zymography, in a range of pH. The relative contribution of protease classes was evaluated with specific inhibitors. [less ▲]